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Emily P Mills,1 Zeynab Alshelh,1 Danny Kosanovic,1 Flavia Di Pietro,1 E Russell Vickers,1 Paul M Macey,2 Luke A Henderson1 1Department of Anatomy and Histology, University of Sydney, Sydney, NSW 2006, Australia; 2School of Nursing and Brain Research Institute, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USACorrespondence: Luke A HendersonDepartment of Anatomy and Histology, F13, University of Sydney, Sydney, NSW, AustraliaTel +612 9351 7063Fax +612 9351 6556Email lukeh@anatomy.usyd.edu.auBackground: Chronic pain, particularly that following nerve injury, can occur in the absence of external stimuli. Although the ongoing pain is sometimes continuous, in many individuals the intensity of their pain fluctuates. Experimental animal studies have shown that the brainstem contains circuits that modulate nociceptive information at the primary afferent synapse and these circuits are involved in maintaining ongoing continuous neuropathic pain. However, it remains unknown if these circuits are involved in regulating fluctuations of ongoing neuropathic pain in humans.Methods: We used functional magnetic resonance imaging to determine whether in 19 subjects with painful trigeminal neuropathy, brainstem pain-modulation circuitry function changes according to moment-to-moment fluctuations in spontaneous pain intensity as rated online over a 12-minute period.Results: We found that when pain intensity was spontaneously high, connectivity strengths between regions of the brainstem endogenous pain-modulating circuitry—the midbrain periaqueductal gray, rostral ventromedial medulla (RVM), and the spinal trigeminal nucleus (SpV)—were high, and vice-versa (when pain was low, connectivity was low). Additionally, sliding-window connectivity analysis using 50-second windows revealed a significant positive relationship between ongoing pain intensity and RVM-SpV connectivity over the duration of the 12-minute scan.Conclusion: These data reveal that moment-to-moment changes in brainstem pain-modulation circuitry functioning likely contribute to fluctuations in spontaneous pain intensity in individuals with chronic neuropathic pain.Keywords: midbrain periaqueductal gray matter, rostral ventromedial medulla, spinal trigeminal nucleus, dynamic connectivity, functional connectivity, spontaneous pain

Zeynab Alshelh,1 Emily P Mills,1 Danny Kosanovic,1 Flavia Di Pietro,1 Paul M Macey,2 E Russell Vickers,1 Luke A Henderson11Department of Anatomy and Histology, University of Sydney, Sydney, NSW, Australia; 2School of Nursing and Brain Research Institute, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USABackground: Chronic neuropathic pain (NP) is a complex disease that results from damage or presumed damage to the somatosensory nervous system. Current treatment regimens are often ineffective. The major impediment in developing effective treatments is our limited understanding of the underlying mechanisms. Preclinical evidence suggests that glial changes are crucial for the development of NP and a recent study reported oscillatory activity differences within the ascending pain pathway at frequencies similar to that of cyclic gliotransmission in NP. Furthermore, there is evidence that glial modifying medications may be effective in treating NP. The aim of this Phase I open-label clinical trial is to determine whether glial modifying medication palmitoylethanolamide (PEA) will reduce NP and whether this is associated with reductions in oscillatory activity within the pain pathway.Methods: We investigated whether 6 weeks of PEA treatment would reduce pain and infra-slow oscillatory activity within the ascending trigeminal pathway in 22 individuals (17 females) with chronic orofacial NP.Results: PEA reduced pain in 16 (73%) of the 22 subjects, 11 subjects showed pain reduction of over 20%. Whilst both the responders and non-responders showed reductions in infra-slow oscillatory activity where orofacial nociceptor afferents terminate in the brainstem, only responders displayed reductions in the thalamus. Furthermore, functional connections between the brainstem and thalamus were altered only in responders.Conclusion: PEA is effective at relieving NP. This reduction is coupled to a reduction in resting oscillations along the ascending pain pathway that are likely driven by rhythmic astrocytic gliotransmission.Keywords: infra-slow oscillations, astrocytes, connectivity, thalamus, imaging

Objective: To determine the replication kinetics and cytopathic effect (CPE) of feline calicivirus (FCV) in feline corneal epithelial cells (FCEC)., Animals Studied: Seven archived FCV isolates and one archived feline herpesvirus type 1 (FHV-1) isolate, previously obtained from eight domestic short hair cats., Procedures: FCV RNA was extracted for sequencing using Illumina MiSeq, to identify three genomically diverse isolates for further testing. Following reference-based assembly, viral genomes were annotated and assessed. Superficial keratectomies were performed to isolate the corneal epithelium of cats and the cells were cultured in vitro. FCEC were infected with the three chosen FCV isolates and one FHV-1 isolate at two different multiplicity of infection ratios (MOIs, 0.1 and 0.01 PFU/cell) and virus titration was assessed at 0, 2, 6, 12, 24, and 48 h post-infection (hpi). Viral identity was confirmed by RT-qPCR., Results: Three genomically diverse FCV isolates were chosen for further assessment in the FCEC model. All infections of FCEC with FCV led to visible CPE, characterized by epithelial cell rounding and detachment from the plate by 24 hpi, while FHV-1 led to visible CPE within 48 hpi. All three of the FCV isolates replicated effectively in FCEC at both 0.1 and 0.01 MOI, with a peak increase in titer approximately 12-24 hpi., Conclusions: The results support the possible role of FCV as a primary pathogen of the feline ocular surface. FCV replicates in FCEC in vitro, leading to profound CPE., (© 2024 American College of Veterinary Ophthalmologists.)

Neuropathic pain (NP) is a prevalent condition often associated with heightened pain responsiveness suggestive of central sensitization. Neuroimaging biomarkers of treatment outcomes may help develop personalized treatment strategies, but white matter (WM) properties have been underexplored for this purpose. Here we assessed whether WM pathways of the default mode network (DMN: medial prefrontal cortex [mPFC], posterior cingulate cortex, and precuneus) and descending pain modulation system (periaqueductal gray [PAG]) are associated with ketamine analgesia and attenuated temporal summation of pain (TSP, reflecting central sensitization) in NP. We used a fixel-based analysis of diffusion-weighted imaging data to evaluate WM microstructure (fiber density [FD]) and macrostructure (fiber bundle cross-section) within the DMN and mPFC-PAG pathways in 70 individuals who underwent magnetic resonance imaging and TSP testing; 35 with NP who underwent ketamine treatment and 35 age- and sex-matched pain-free individuals. Individuals with NP were assessed before and 1 month after treatment; those with ≥30% pain relief were considered responders (n = 18), or otherwise as nonresponders (n = 17). We found that WM structure within the DMN and mPFC-PAG pathways did not differentiate responders from nonresponders. However, pretreatment FD in the anterior limb of the internal capsule correlated with pain relief (r=.48). Moreover, pretreatment FD in the DMN (left mPFC-precuneus/posterior cingulate cortex; r=.52) and mPFC-PAG (r=.42) negatively correlated with changes in TSP. This suggests that WM microstructure in the DMN and mPFC-PAG pathway is associated with the degree to which ketamine reduces central sensitization. Thus, fixel metrics of WM structure may hold promise to predict ketamine NP treatment outcomes. PERSPECTIVE: We used advanced fixel-based analyses of MRI diffusion-weighted imaging data to identify pretreatment WM microstructure associated with ketamine outcomes, including analgesia and markers of attenuated central sensitization. Exploring associations between brain structure and treatment outcomes could contribute to a personalized approach to treatment for individuals with NP., (Copyright © 2024 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Chronic neuropathic pain can result from nervous system injury and can persist in the absence of external stimuli. Although ongoing pain characterizes the disorder, in many individuals, the intensity of this ongoing pain fluctuates dramatically. Previously, it was identified that functional magnetic resonance imaging signal covariations between the midbrain periaqueductal gray (PAG) matter, rostral ventromedial medulla (RVM), and spinal trigeminal nucleus are associated with moment-to-moment fluctuations in pain intensity in individuals with painful trigeminal neuropathy (PTN). Since this brainstem circuit is modulated by higher brain input, we sought to determine which cortical sites might be influencing this brainstem network during spontaneous fluctuations in pain intensity. Over 12 min, we recorded the ongoing pain intensity in 24 PTN participants and classified them as fluctuating ( n  = 13) or stable ( n  = 11). Using a PAG seed, we identified connections between the PAG and emotional-affective sites such as the hippocampal and posterior cingulate cortices, the sensory-discriminative posterior insula, and cognitive-affective sites such as the dorsolateral prefrontal (dlPFC) and subgenual anterior cingulate cortices that were altered dependent on spontaneous high and low pain intensity. Additionally, sliding-window functional connectivity analysis revealed that the dlPFC-PAG connection anticorrelated with perceived pain intensity over the entire 12 min period. These findings reveal cortical systems underlying moment-to-moment changes in perceived pain in PTN, which likely cause dysregulation in the brainstem circuits previously identified, and consequently alter the appraisal of pain across time., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Meylakh et al.)

Objective: Feline herpesvirus 1 (FHV-1) causes ocular surface disease in domestic cats. The purpose of this study was to assess the relationship between bacterial ocular surface microbiota and outcomes for cats with FHV-1 ocular surface disease., Animals Studied: Twenty-two shelter-housed cats with confirmed FHV-1 ocular surface disease., Procedures: Animals were grouped according to FHV-1 shedding and ocular clinical scores following intervention: worsened outcome (WorOut, n = 11) or improved outcome (ImpOut, n = 11). Scoring and conjunctival sampling were completed on Days 1 and 8 of twice daily antiviral treatment. Bacterial DNA was extracted and submitted for 16S rRNA gene sequencing. Real-time polymerase chain reaction was performed for selected bacterial species. Overall DNA concentration between groups was assessed., Results: Bacterial microbiota relative abundance composition was significantly different between ImpOut and WorOut groups (weighted UniFrac p = .006). Alpha diversity was significantly higher in the ImpOut group compared with the WorOut group (Shannon p = .042, Simpson's p = .022, Pielou's p = .037). Differences in the relative abundance of various phyla and species were detected between groups. Total DNA concentration was higher in the WorOut group compared with the ImpOut group (p = .04). Feline GAPDH (p = .001) and Bilophila wadsworthia (p = .024) copy number was significantly higher in the ImpOut group compared with the WorOut group., Conclusions: The results highlight the important relationship between the bacterial ocular surface microbiota and FHV-1 infection outcomes in cats treated with antiviral medications. Low bacterial species diversity, higher overall DNA (presumed predominantly bacterial) load, and certain bacterial phyla/species were associated with poor outcomes for cats with FHV-1 ocular disease., (© 2023 American College of Veterinary Ophthalmologists.)

Background: Equid alphaherpesvirus 1 (EHV-1) is a global viral pathogen of domestic equids which causes reproductive, respiratory and neurological disease. Few isolates acquired from naturally infected USA-based hosts have been fully sequenced and analyzed to date. An ORF 30 (DNA polymerase) variant (A2254G) has previously been associated with neurological disease in host animals. The purpose of this study was to perform phylogenomic analysis of EHV-1 isolates acquired from USA-based hosts and compare these isolates to previously sequenced global isolates., Methods: EHV-1 was isolated from 23 naturally infected USA-based equids (6 different states, 15 disease outbreaks) with reproductive (22/23) or neurological disease (1/23). Following virus isolation, EHV-1 DNA was extracted for sequencing using Illumina MiSeq. Following reference-based assembly, whole viral genomes were annotated and assessed. Previously sequenced EHV-1 isolates (n = 114) obtained from global host equids were included in phylogenomic analyses., Results: The overall average genomic distance was 0.0828% (SE 0.004%) for the 23 newly sequenced USA isolates and 0.0705% (SE 0.003%) when all 137 isolates were included. Clade structure was predominantly based on geographic origin. Numerous nucleotide substitutions (mean [range], 179 [114-297] synonymous and 81 [38-120] non-synonymous substitutions per isolate) were identified throughout the genome of the newly sequenced USA isolates. The previously described ORF 30 A2254G substitution (associated with neurological disease) was found in only one isolate obtained from a host with non-neurological clinical signs (reproductive disease), six additional, unique, non-synonymous ORF 30 substitutions were detected in 22/23 USA isolates. Evidence of recombination was present in most (22/23) of the newly sequenced USA isolates., Conclusions: Overall, the genomes of the 23 newly sequenced EHV-1 isolates obtained from USA-based hosts were broadly similar to global isolates. The previously described ORF 30 A2254G neurological substitution was infrequently detected in the newly sequenced USA isolates, most of which were obtained from host animals with reproductive disease. Recombination was likely to be partially responsible for genomic diversity in the newly sequenced USA isolates., (© 2023. The Author(s).)

Objective: The purpose of this study was to validate the use of the Reichert Tono-Vera® Vet tonometer rabbit setting in normal ex vivo rabbit eyes and to compare the rabbit setting to the dog, cat, and horse settings of this tonometer., Procedure: Six freshly enucleated normal rabbit eyes were cannulated and connected to a fluid reservoir and physiologic monitor. Triplicate measurements were obtained with the four available settings: dog, cat, horse, and rabbit at various intraocular pressures (IOP) ranging from 5 to 80 mmHg. Bland-Altman analysis was utilized to determine bias and 95% limits of agreement for each setting., Results: Linear regression equations for the dog, horse, cat, and rabbit settings were y = 0.8101x + 2.5058, y = 0.7594x - 3.4673, y = 0.6635x + 0.3021, and y = 0.8935x + 1.3295, respectively. All settings demonstrated strong positive linear trends (dog r 2  = 0.9644, horse r 2  = 0.9456, cat r 2  = 0.9309, and rabbit r 2  = 0.9558). Bland-Altman plots revealed that the average bias and 95% limits of agreement (mmHg) were -4.73, -12.65, -12.86 and -2.73 and (-15.31, 5.86), (-29.03, 3.74), (-25.67, -0.05), and (-12.21, 6.76) for the dog, horse, cat, and rabbit settings, respectively., Conclusion: The Tono-Vera® Vet rabbit setting provided the most accurate and precise measurements compared with the other settings, but slightly underestimated actual IOP, especially as IOP was increased. This tonometer, using the rabbit setting, is likely to be appropriate for the estimation of IOP in rabbits with the appropriate correction formula applied., (© 2023 American College of Veterinary Ophthalmologists.)

Prior experiences, conditioning cues, and expectations of improvement are essential for placebo analgesia expression. The dorsolateral prefrontal cortex is considered a key region for converting these factors into placebo responses. Since dorsolateral prefrontal cortex neuromodulation can attenuate or amplify placebo, we sought to investigate dorsolateral prefrontal cortex biochemistry and function in 38 healthy individuals during placebo analgesia. After conditioning participants to expect pain relief from a placebo "lidocaine" cream, we collected baseline magnetic resonance spectroscopy (1H-MRS) at 7 Tesla over the right dorsolateral prefrontal cortex. Following this, functional magnetic resonance imaging scans were collected during which identical noxious heat stimuli were delivered to the control and placebo-treated forearm sites. There was no significant difference in the concentration of gamma-aminobutyric acid, glutamate, Myo-inositol, or N-acetylaspartate at the level of the right dorsolateral prefrontal cortex between placebo responders and nonresponders. However, we identified a significant inverse relationship between the excitatory neurotransmitter glutamate and pain rating variability during conditioning. Moreover, we found placebo-related activation within the right dorsolateral prefrontal cortex and altered functional magnetic resonance imaging coupling between the dorsolateral prefrontal cortex and the midbrain periaqueductal gray, which also correlated with dorsolateral prefrontal cortex glutamate. These data suggest that the dorsolateral prefrontal cortex formulates stimulus-response relationships during conditioning, which are then translated to altered cortico-brainstem functional relationships and placebo analgesia expression., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Feline herpesvirus type 1 (FHV-1) commonly causes ocular surface disease in cats and is treated with antiviral medications targeting viral DNA polymerase (UL30/42). Herein, we describe a method to assess the FHV-1 genome for mutation development and to assess the functional impact of mutations, if present. Fourteen shelter-housed domestic cats with FHV-1 ocular surface disease were assigned to one of four treatment groups: placebo ( n  = 3), cidofovir 0.5% ophthalmic solution ( n  = 3), famciclovir oral solution ( n  = 5), or ganciclovir 0.15% ophthalmic solution ( n  = 3). Swabs were collected before (day 1) and after (day 8) 1 week of twice-daily treatments to isolate viable FHV-1. Viral DNA was extracted for sequencing using Illumina MiSeq with subsequent genomic variant detection between paired day 1 and day 8 isolates. Plaque reduction assay was performed on paired isolates demonstrating non-synonymous variants. A total of 171 synonymous and 3 non-synonymous variants were identified in day 8 isolates. No variants were detected in viral UL23, UL30, or UL42 genes. Variant totals were not statistically different in animals receiving antiviral or placebo ( p  = 0.4997). A day 8 isolate from each antiviral treatment group contained a single non-synonymous variant in ICP4 (transcriptional regulator). These 3 isolates demonstrated no evidence of functional antiviral resistance when IC 50 was assessed. Most (10/14 pairs) day 1 and 8 viral isolate pairs from the same host animal were near-identical. While functional variants were not detected in this small sample, these techniques can be replicated to assess FHV-1 isolates suspected of having developed resistance to antiviral medications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lewin, Ineck, Mironovich, Marino, Liu, Emelogu, Mills, Camacho-Luna and Carter.)

Objective: Porcine models of ocular disease are becoming increasingly utilized. A recently commercialized ocular tonometer, the Reichert Tono-Vera® Vet, has not been evaluated for use in pigs. The purpose of this study was to calibrate this device for use in porcine eyes and to determine which settings are most appropriate for use in pigs., Procedure: The anterior chambers of five freshly enucleated normal porcine eyes were cannulated then connected to a reservoir of balanced salt solution and a physiologic monitor. Triplicate measurements were obtained with the four available settings: dog, cat, horse, and rabbit at intraocular pressures ranging from 5- to 80 mmHg. Bland-Altman analysis was utilized to determine bias and 95% limits of agreement for each setting., Results: There was a strong positive linear regression trend for all settings (dog r 2  = 0.986, horse r 2  = 0.947, cat r 2  = 0.977, and rabbit r 2  = 0.982). The linear regression equations for the dog, horse, cat, and rabbit setting were y = 1.0168x - 2.6128, y = 0.8743x - 3.4959, y = 0.9394x - 7.3188, and y = 1.1082x - 3.4077. The average bias and 95% limits of agreement for dog, horse, cat, and rabbit settings were - 2.00, -8.32, -9.58, and 0.57 mmHg, and (-7.52, 3.53), (-19.00, 2.37), (-16.66, -2.50), and (-7.79, 8.93), in mmHg., Conclusion: The Tono-Vera® Vet dog setting was most accurate and precise setting compared to true intraocular pressures. This setting is likely to be appropriate for in vivo use in pigs, with the appropriate correction formula applied., (© 2023 American College of Veterinary Ophthalmologists.)

Background: Pupillometry, the measurement of pupil size and reactivity to a stimulus, has various uses in both human and veterinary medicine. These reflect autonomic tone, with the potential to assess nociception and emotion. Infrared pupillometry reduces inaccuracies that may occur when the pupillary light reflex is determined subjectively by the examiner. To our knowledge, there are no published studies outlining normal reference intervals for automated pupillometry in dogs., Objective: The objective of this study was to develop de novo automated pupillometry reference intervals from 126 healthy canine eyes., Methods: The pupillary light reflex (PLR) was measured with a handheld pupillometer (NeurOptics™ PLR-200™ Pupillometer). Parameters recorded included maximum pupil diameter (MAX), minimum pupil diameter (MIN), percent constriction (CON), latency (LAT), average constriction velocity (ACV), maximum constriction velocity (MCV), average dilation velocity (ADV) and time to 75% pupil diameter recovery (T75). One measurement was obtained for each eye., Results: The following reference intervals were developed: MAX (6.05-11.30 mm), MIN (3.76-9.44 mm), CON (-37.89 to -9.64 %), LAT (0.11-0.30 s), ACV (-6.39 to -2.63 mm/ s), MCV (-8.45 to -3.75 mm/s), ADV (-0.21-1.77 mm/s), and T75 (0.49-3.20 s)., Clinical Significance: The reference intervals developed in this study are an essential first step to facilitate future research exploring pupillometry as a pain assessment method in dogs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mills, Combs-Ramey, Kwong and Pang.)

Conditioned pain modulation (CPM) is a physiological measure thought to reflect an individual's endogenous pain modulation system. CPM varies across individuals and provides insight into chronic pain pathophysiology. There is growing evidence that CPM may help predict individual pain treatment outcome. However, paradigm variabilities and practical issues have impeded widespread clinical adoption of CPM assessment. This study aimed to compare two CPM paradigms in people with chronic pain and healthy individuals. A total of 30 individuals (12 chronic pain, 18 healthy) underwent two CPM paradigms. The heat CPM paradigm acquired pain intensity ratings evoked by a test stimulus (TS) applied before and during the conditioning stimulus (CS). The pressure CPM paradigm acquired continuous pain intensity ratings of a gradually increasing TS, before and during CS. Pain intensity was rated from 0 (no pain) to 100 (worst pain imaginable); Pain50 is the stimulus level for a response rated 50. Heat and pressure CPM were calculated as a change in TS pain intensity ratings at Pain50, where negative CPM scores indicate pain inhibition. We also determined CPM in the pressure paradigm as change in pressure pain detection threshold (PDT). We found that in healthy individuals the CPM effect was significantly more inhibitory using the pressure paradigm than the heat paradigm. The pressure CPM effect was also significantly more inhibitory when based on changes at Pain50 than at PDT. However, in individuals with chronic pain there was no significant difference in pressure CPM compared to heat or PDT CPM. There was no significant correlation between clinical pain measures (painDETECT and Brief Pain Inventory) and paradigm type (heat vs. pressure), although heat-based CPM and painDETECT scores showed a trend. Importantly, the pressure paradigm could be administered in less time than the heat paradigm. Thus, our study indicates that in healthy individuals, interpretation of CPM findings should consider potential modality-dependent effects. However, in individuals with chronic pain, either heat or pressure paradigms can similarly be used to assess CPM. Given the practical advantages of the pressure paradigm (e.g., short test time, ease of use), we propose this approach to be well-suited for clinical adoption., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 El-Sayed, Fauchon, Kim, Firouzian, Osborne, Besik, Mills, Bhatia and Davis.)

Pain perception can be powerfully influenced by an individual's expectations and beliefs. Although the cortical circuitry responsible for pain modulation has been thoroughly investigated, the brainstem pathways involved in the modulatory phenomena of placebo analgesia and nocebo hyperalgesia remain to be directly addressed. This study used ultra-high-field 7 tesla functional MRI (fMRI) to accurately resolve differences in brainstem circuitry present during the generation of placebo analgesia and nocebo hyperalgesia in healthy human participants ( N = 25, 12 male). Over 2 successive days, through blinded application of altered thermal stimuli, participants were deceptively conditioned to believe that two inert creams labeled lidocaine (placebo) and capsaicin (nocebo) were acting to modulate their pain relative to a third Vaseline (control) cream. In a subsequent test phase, fMRI image sets were collected while participants were given identical noxious stimuli to all three cream sites. Pain intensity ratings were collected and placebo and nocebo responses determined. Brainstem-specific fMRI analysis revealed altered activity in key pain modulatory nuclei, including a disparate recruitment of the periaqueductal gray (PAG)-rostral ventromedial medulla (RVM) pathway when both greater placebo and nocebo effects were observed. Additionally, we found that placebo and nocebo responses differentially activated the parabrachial nucleus but overlapped in engagement of the substantia nigra and locus coeruleus. These data reveal that placebo and nocebo effects are generated through differential engagement of the PAG-RVM pathway, which in concert with other brainstem sites likely influences the experience of pain by modulating activity at the level of the dorsal horn. SIGNIFICANCE STATEMENT Understanding endogenous pain modulatory mechanisms would support development of effective clinical treatment strategies for both acute and chronic pain. Specific brainstem nuclei have long been known to play a central role in nociceptive modulation; however, because of the small size and complex organization of the nuclei, previous neuroimaging efforts have been limited in directly identifying how these subcortical networks interact during the development of antinociceptive and pro-nociceptive effects. We used ultra-high-field fMRI to resolve brainstem structures and measure signal change during placebo analgesia and nocebo hyperalgesia. We define overlapping and disparate brainstem circuitry responsible for altering pain perception. These findings extend our understanding of the detailed organization and function of discrete brainstem nuclei involved in pain processing and modulation., (Copyright © 2021 the authors.)

Acute pain serves as a protective mechanism that alerts us to potential tissue damage and drives a behavioural response that removes us from danger. The neural circuitry critical for mounting this behavioural response is situated within the brainstem and is also crucial for producing analgesic and hyperalgesic responses. In particular, the periaqueductal grey, rostral ventromedial medulla, locus coeruleus and subnucleus reticularis dorsalis are important structures that directly or indirectly modulate nociceptive transmission at the primary nociceptive synapse. Substantial evidence from experimental animal studies suggests that plasticity within this system contributes to the initiation and/or maintenance of chronic neuropathic pain, and may even predispose individuals to developing chronic pain. Indeed, overwhelming evidence indicates that plasticity within this circuitry favours pro-nociception at the primary synapse in neuropathic pain conditions, a process that ultimately contributes to a hyperalgesic state. Although experimental animal investigations have been crucial in our understanding of the anatomy and function of the brainstem pain-modulation circuitry, it is vital to understand this system in acute and chronic pain states in humans so that more effective treatments can be developed. Recent functional MRI studies have identified a key role of this system during various analgesic and hyperalgesic responses including placebo analgesia, offset analgesia, attentional analgesia, conditioned pain modulation, central sensitisation and temporal summation. Moreover, recent MRI investigations have begun to explore brainstem pain-modulation circuitry plasticity in chronic neuropathic pain conditions and have identified altered grey matter volumes and functioning throughout the circuitry. Considering the findings from animal investigations, it is likely that these changes reflect a shift towards pro-nociception that ultimately contributes to the maintenance of neuropathic pain. The purpose of this review is to provide an overview of the human brain imaging investigations that have improved our understanding of the pain-modulation system in acute pain states and in neuropathic conditions. Our interpretation of the findings from these studies is often guided by the existing body of experimental animal literature, in addition to evidence from psychophysical investigations. Overall, understanding the plasticity of this system in human neuropathic pain conditions alongside the existing experimental animal literature will ultimately improve treatment options., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mills, Keay and Henderson.)

There is evidence from preclinical models of chronic pain and human psychophysical investigations to suggest that alterations in endogenous brainstem pain-modulation circuit functioning are critical for the initiation and/or maintenance of pain. Whilst preclinical models have begun to explore the functioning of this circuitry in chronic pain, little is known about such functioning in humans with chronic pain. The aim of this investigation was to determine whether individuals with chronic non-neuropathic pain, painful temporomandibular disorders (TMD), display alterations in brainstem pain-modulating circuits. Using resting-state functional magnetic resonance imaging, we performed static and dynamic functional connectivity (FC) analyses to assess ongoing circuit function in 16 TMD and 45 control subjects. We calculated static FC as the correlation of functional magnetic resonance imaging signals between regions over the entire scan and dynamic FC as the correlation of signals in short (50s) windows. Compared with controls, TMD subjects showed significantly greater (static) FC between the rostral ventromedial medulla and both the subnucleus reticularis dorsalis and the region that receives orofacial nociceptive afferents, the spinal trigeminal nucleus. No differences were found in other brainstem pain-modulating regions such as the midbrain periaqueductal gray matter and locus coeruleus. We also identified that TMD subjects experience greater variability in the dynamic functional connections between the rostral ventromedial medulla and both the subnucleus reticularis dorsalis and spinal trigeminal nucleus. These changes may underlie enhanced descending pain-facilitating actions over the region that receives nociceptive afferents, ultimately leading to enhanced nociceptive transmission to higher brain regions and thus contributing to the ongoing perception of pain. PERSPECTIVE: Psychophysical studies suggest that brainstem pain-modulation circuits contribute to the maintenance of chronic pain. We report that individuals with painful TMD display altered static and dynamic FC within the brainstem pain-modulation network. Modifying this circuitry may alter an individual's ongoing pain., (Copyright © 2020 United States Association for the Study of Pain, Inc. All rights reserved.)

Pain is a complex phenomenon that is highly modifiable by expectation. Whilst the intensity of incoming noxious information plays a key role in the intensity of perceived pain, this intensity can be profoundly shaped by an individual's expectations. Modern brain imaging investigations have begun to detail the brain regions responsible for placebo and nocebo related changes in pain, but less is known about the neural basis of stimulus-expectancy changes in pain processing. In this functional magnetic resonance imaging study, we administered two separate protocols of the same noxious thermal stimuli to 24 healthy subjects. However, different expectations were elicited by different explanations to subjects prior to each protocol. During one protocol, pain intensities were matched to expectation and in the other protocol they were not. Pain intensity was measured continuously via a manually operated computerized visual analogue scale. When individuals expected the stimulus intensity to remain constant, but in reality it was surreptitiously increased or decreased, pain intensity ratings were significantly lower than when expectation and pain intensities were matched. When the stimulus intensities did not match expectations, various areas in the brain such as the amygdala, anterior cingulate cortex (ACC), dorsolateral prefrontal cortex (dlPFC), and the midbrain periaqueductal gray matter (PAG) displayed significantly different patterns of activity compared to instances when stimulus intensity and pain expectations were matched. These results show that stimulus-expectancy manipulation of pain intensity alters activity in both higher brain and brainstem centers which are known to modulate pain under various conditions., (Copyright © 2020 Henderson, Di Pietro, Youssef, Lee, Tam, Akhter, Mills, Murray, Peck and Macey.)

The neural mechanism responsible for migraine remains unclear. While an external trigger has been proposed to initiate a migraine, it has also been proposed that changes in brainstem function are critical for migraine headache initiation and maintenance. Although the idea of altered brainstem function has some indirect support, no study has directly measured brainstem pain modulation circuitry function in migraineurs particularly immediately before a migraine. In male and female humans, we performed fMRI in 31 controls and 31 migraineurs at various times in their migraine cycle. We measured brainstem function during noxious orofacial stimulation and assessed resting-state functional connectivity. First, we found that, in individual migraineurs, pain sensitivity increased over the interictal period but then dramatically decreased immediately before a migraine. Second, despite overall similar pain intensity ratings between groups, in the period immediately before a migraine, compared with controls and other migraine phases, migraineurs displayed greater activation in the spinal trigeminal nucleus during noxious orofacial stimulation and reduced functional connectivity of this region with the rostral ventromedial medulla. Additionally, during the interictal phase, migraineurs displayed reduced activation of the midbrain periaqueductal gray matter and enhanced periaqueductal gray connectivity with the rostral ventromedial medulla. These data support the hypothesis that brainstem sensitivity fluctuates throughout the migraine cycle. However, in contrast to the prevailing hypothesis, our data suggest that, immediately before a migraine attack, endogenous analgesic mechanisms are enhanced and incoming noxious inputs are less likely to reach higher brain centers. SIGNIFICANCE STATEMENT It has been hypothesized that alterations in brainstem function are critical for the generation of migraine. In particular, modulation of orofacial pain pathways by brainstem circuits alters the propensity of external triggers or ongoing spontaneous activity to evoke a migraine attack. We sought to obtain empirical evidence to support this theory. Contrary to our hypothesis, we found that pain sensitivity decreased immediately before a migraine, and this was coupled with increased sensitivity of the spinal trigeminal nucleus to noxious stimuli. We also found that resting connectivity within endogenous pain modulation circuitry alters across the migraine cycle. These changes may reflect enhanced and diminished neural tone states proposed to be critical for the generation of a migraine and underlie cyclic fluctuations in migraine brainstem sensitivity., (Copyright © 2018 the authors 0270-6474/18/3810479-10$15.00/0.)

The neural mechanisms underlying the development and maintenance of chronic pain following nerve injury remain unclear. There is growing evidence that chronic neuropathic pain is associated with altered thalamic firing patterns, thalamocortical dysrhythmia and altered infra-slow oscillations in ascending pain pathways. Preclinical and post-mortem human studies have revealed that neuropathic pain is associated with prolonged astrocyte activation in the dorsal horn and we have suggested that this may result in altered gliotransmission, which results in altered resting neural rhythm in the ascending pain pathway. Evidence of astrocyte activation above the level of the dorsal horn in living humans is lacking and direct measurement of astrocyte activation in living humans is not possible, however, there is evidence that regional alterations in T2 relaxation times are indicative of astrogliosis. The aim of this study was to use T2 relaxometry to explore regional brain anatomy of the ascending pain pathway in individuals with chronic orofacial neuropathic pain. We found that in individuals with trigeminal neuropathic pain, decreases in T2 relaxation times occurred in the region of the spinal trigeminal nucleus and primary somatosensory cortex, as well as in higher order processing regions such as the dorsolateral prefrontal, cingulate and hippocampal/parahippocampal cortices. We speculate that these regional changes in T2 relaxation times reflect prolonged astrocyte activation, which results in altered brain rhythm and ultimately the constant perception of pain. Blocking prolonged astrocyte activation may be effective in preventing and even reversing the development of chronic pain following neural injury.

Preclinical investigations have suggested that altered functioning of brainstem pain-modulation circuits may be crucial for the maintenance of some chronic pain conditions. While some human psychophysical studies show that patients with chronic pain display altered pain-modulation efficacy, it remains unknown whether brainstem pain-modulation circuits are altered in individuals with chronic pain. The aim of the present investigation was to determine whether, in humans, chronic pain following nerve injury is associated with altered ongoing functioning of the brainstem descending modulation systems. Using resting-state functional magnetic resonance imaging, we found that male and female patients with chronic neuropathic orofacial pain show increased functional connectivity between the rostral ventromedial medulla (RVM) and other brainstem pain-modulatory regions, including the ventrolateral periaqueductal gray (vlPAG) and locus ceruleus (LC). We also identified an increase in RVM functional connectivity with the region that receives orofacial nociceptor afferents, the spinal trigeminal nucleus. In addition, the vlPAG and LC displayed increased functional connectivity strengths with higher brain regions, including the hippocampus, nucleus accumbens, and anterior cingulate cortex, in individuals with chronic pain. These data reveal that chronic pain is associated with altered ongoing functioning within the endogenous pain-modulation network. These changes may underlie enhanced descending facilitation of processing at the primary synapse, resulting in increased nociceptive transmission to higher brain centers. Further, our findings show that higher brain regions interact with the brainstem modulation system differently in chronic pain, possibly reflecting top-down engagement of the circuitry alongside altered reward processing in pain conditions. SIGNIFICANCE STATEMENT Experimental animal models and human psychophysical studies suggest that altered functioning of brainstem pain-modulation systems contributes to the maintenance of chronic pain. However, the function of this circuitry has not yet been explored in humans with chronic pain. In this study, we report that individuals with orofacial neuropathic pain show altered functional connectivity between regions within the brainstem pain-modulation network. We suggest that these changes reflect largely central mechanisms that feed back onto the primary nociceptive synapse and enhance the transfer of noxious information to higher brain regions, thus contributing to the constant perception of pain. Identifying the mechanisms responsible for the maintenance of neuropathic pain is imperative for the development of more efficacious therapies., (Copyright © 2018 the authors 0270-6474/18/380465-09$15.00/0.)

Objective: To review current literature for the efficacy and safety of treatment for nonalcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM)., Data Sources: A PubMed literature search from January 1990 to June 2017 was conducted using the search terms nonalcoholic fatty liver disease, diabetes mellitus, type 2, therapy, treatment, treat, therapeutics, nonalcoholic fatty liver, nonalcoholic hepatosteatosis, NASH, NAFLD, metformin, and statin. Bibliographies of chosen articles were reviewed., Study Selection and Data Extraction: Relevant articles on metformin, thiazolidinediones (TZD), glucagon-like peptide-1 receptor agonists (GLP-1 RA), and statins for the treatment of NAFLD which included patients with T2DM were reviewed. A total of 23 relevant studies were found and included randomized controlled, observational, and open-label designs, as well as three meta-analyses., Data Synthesis: Metformin combined with weight loss provides a modest improvement in steatosis and no improvement in fibrosis in patients with NAFLD and T2DM. TZDs showed positive results on fibrosis and resolution of NASH but at least half of patients studied were nonresponders. GLP-1 RAs also showed favorable results on reductions in transaminases and steatosis and improvements in insulin sensitivity and weight loss but lack efficacy data for resolution of NASH or improvement in fibrosis scores. Statins showed favorable results on reductions in transaminases but mixed results for improvement in steatosis and fibrosis scores., Conclusion: All reviewed treatment options are safe for management of NAFLD in patients with T2DM but long-term histological improvements are minimal. TZDs are efficacious for resolution of NASH and improvements in fibrosis but long-term use is required to maintain these results., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest.

It has been proposed that pain competes with other attention-demanding stimuli for cognitive resources, and many chronic pain patients display significant attention and mental flexibility deficits. These alterations may result from disruptions in the functioning of the default mode network (DMN) which plays a critical role in attention, memory, prospection and self-processing, and recent investigations have found alterations in DMN function in multiple chronic pain conditions. Whilst it has been proposed that these DMN alterations are a characteristic of pain that is chronic in nature, we recently reported altered oscillatory activity in the DMN during an acute, 5  minute noxious stimulus in healthy control subjects. We therefore hypothesize that altered DMN activity patterns will not be restricted to those in chronic pain but instead will also occur in healthy individuals during tonic noxious stimuli. We used functional magnetic resonance imaging to measure resting state infra-slow oscillatory activity and functional connectivity in patients with chronic orofacial pain at rest and in healthy controls during a 20-minute tonic pain stimulus. We found decreases in oscillatory activity in key regions of the DMN in patients with chronic pain, as well as in healthy controls during tonic pain in addition to changes in functional connectivity between the posterior cingulate cortex and areas of the DMN in both groups. The results show that similar alterations in DMN function occur in healthy individuals during acute noxious stimuli as well as in individuals with chronic pain. These DMN changes may reflect the presence of pain per se and may underlie alterations in attentional processes that occur in the presence of pain.

Postnatal depression (PND) has been underreported in South Africa. This retrospective study investigated factors which appear to predispose women to PND. Two groups, one consisting of women who suffered from PND and the other of women free of this complaint, provided information on a number of biological, psychological and social factors. In line with current opinion it was found that no single causative factor could be isolated but that a variety of factors may contribute to the problem. Among the factors which distinguished the two groups were the mothers' emotional health during pregnancy, complications after birth, marital relations, relationship with their own mothers, social support and preparation for motherhood. An alarming finding was that a large proportion of the PND sufferers had not known of the disorder's existence before their own diagnosis.